This proposal focuses on the mechanism and pathway of prohead assembly with bacteriophage phi29 as a model. The key structural proteins found in phi29 prohead are the scaffolding (gp7), major capsid (gp8) and the portal vertex (gp10). Prohead assembly will be studied in vitro with the purified gp7, gp8 and gp10. Considering the involvement of host factors and functions in prohead assembly, extract complementation studies with E. coli expressing or co-expressing the three structural proteins will be performed as pilot experiments. The prohead assembly pathway will be elucidated through the use of electron microscopy and electrophoresis. To assay for biological activity, the highly efficient in vitro packaging system will be used. The sequential interactions of the structural proteins will be studied by: a) complementation with E. coli extracts, one of which contains two prohead components as potential assembly intermediate; b) in vitro assembly with purified prohead components and assembly intermediates; c) Pulse labelling in vivo with E. coli co-expressing gp7, gp8 and gpl0. Complexes of gp7-gplO and, possibly, gp7-gp8 oligomers will be isolated and mixed with gp8 and gp 10, respectively, to study their interactions and the formation of particles. The mechanism of prohead size and shape determination will be studied by the following approaches: a) construction of chimeric phi29 proheads with portal vertexes of phages T4 or T3, and scaffolding proteins of lambda or T3; b) sequencing of gp7 and gpl0 genes of phi29 suppressor-sensitive or ts mutants which produce proheads with variable shape and size, comparison of the sequences with wild type phi29 DNA, and localization of the mutation: c) introduction of mutations into the gp7 and gp 10 genes by in vitro mutagenesis; d) construction of proheads containing dimeric scaffolding proteins with N- to C-terminus linkage. The mechanism and the structural basis for the six-fold/five-fold mismatch between the head and the portal vertex will be studied by analyzing the scaffold/portal vertex complex and the sRNA/portal vertex decamer with three dimensional image analysis procedures. With the in vitro mutagenesis, DNA deletion and insertion techniques, the domains for the scaffold/portal vertex interaction will be identified. Overall, the objective of this study is to elucidate the fundamental mechanism of prohead morphogenesis. In terms of practical applications, this study could provide information for: a) construction of viral vectors for gene cloning, b) development of synthetic viral particles as vaccines for human and animal non-enveloped viruses, such as papilloma virus, based on the viral structure, and c) design of anti-viral drugs that interact with specific viral targets with little or no toxicity to the host cells.